Sensitive, selective, and irreversible inhibition of cyclooxygenase-2 activity by copper.
Sensitive, selective, and irreversible inhibition of cyclooxygenase-2 activity by copper.
复制标题
铜对 cyclooxygenase-2 活性具有灵敏、选择性和不可逆的抑制作用。
DOI：
10.1002/cmdc.200700217
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发表时间：
2008
期刊：
影响因子：
3.4
通讯作者：
Bush,AshleyI
中科院分区：
文献类型：
--
作者：
Nagano,Seiichi;Bush,AshleyI
环氧合酶(COX)是一种前炎性酶，催化限速反应从花生四烯酸合成许多重要的前列腺素。COX-1是一种结构型，广泛表达于各种组织中，为前列腺素的基础生产服务。COX-2是一种受炎症介质和生长因子如白介素1和肿瘤生长因子β上调的诱导型。COX通过两个连续的催化活性产生前列腺素H2(PGH2)。花生四烯酸首先通过真正的环氧合酶活性转化为前列腺素G2(PGG2)，然后通过酶的过氧化物酶活性将PGG2还原为PGH2。PGH2进一步转化为前列腺素E2(PGE2)，这是一种具有血小板聚集等生理功能的前列腺素。[1]COX在活性部位有一个亚铁血红素，亚铁血红素的氧化还原状态对该酶的环氧合酶和过氧化物酶活性至关重要。[2]然而，环氧合酶容易自我失活，这可能是因为该酶的亚铁血红素活性部位的酪氨酸残基被氧化。[3]因此，COX容易通过酶的氧化修饰而失去活性。据报道，COX-2在炎症性疾病中的表达增加，如类风湿性关节炎(RA)，[4，5]和神经退行性疾病如肌萎缩侧索硬化症[6]和阿尔茨海默病(AD)[7]，在这些疾病中，这种酶与β-淀粉样蛋白氧化交联。[8]因此，COX-2的典型抑制剂非类固醇抗炎药(NSAID)可能对这些疾病有有益的影响。[9-11]有证据表明，Cu2+的异常新陈代谢也可能在这些疾病中起致病作用。我们报道了COX-2被生理浓度的Cu2+离子特异性地灭活，并讨论了它在临床情况下的相关性。表1显示了各种生物金属对环氧合酶-2活性产生前列腺素E_2的影响。我们测量了PGE2的产量以反映COX-2的活性，因为PGE2是部分纯化的酶的主要最终产物[17]，可以用EIA灵敏地定量。我们没有从不含COX-2的样本中发现任何PGE2的产生(数据未显示)。在500 nm处，只有Cu2+对PGE2的产生有显著抑制作用(P<；0.01，t检验)，抑制率为50%。Cu2+的配体(3μM甘氨酸)本身对该酶没有抑制作用(数据未显示)。我们缓冲液中Cu2+的本底水平通常测量在20 nm以下，[18]与本研究中添加的Cu2+浓度相比，这被认为是微不足道的。Cu2+以浓度依赖的方式抑制COX-2活性(图1)。Cu2+的IC50为%500 nm，其化学计量比为3.5：1(Cu2+：酶)。另一方面，在10μM EDTA存在下，Cu2+在浓度达2000 nM时的抑制作用被消除(图1)。在不含Cu2+的EDTA存在下，PGE_2的产生量低于不含EDTA的样品。这可能是因为EDTA从COX-2的活性部位将一些铁螯合到血红素上，从而降低了酶的活性。
Cyclooxygenase (COX) is a proinflammatory enzyme that catalyzes the rate-limiting reaction to produce many important prostaglandins from arachidonic acid.[1] COX has two isoforms. COX-1 is the constitutive type, widely expressed in various tissues, that serves the basal production of prostaglandins. COX-2 is the inducible type that is upregulated by inflammatory mediators and growth factors such as interleukin-1 and tumor growth factor β. COX produces prostaglandin H2 (PGH2) by two consecutive catalytic activities. Arachidonic acid is converted to prostaglandin G2 (PGG2) by the authentic “cyclooxygenase” activity at first, and then PGG2 is reduced to PGH2 by the peroxidase activity of the enzyme. PGH2 is further converted to prostaglandin E2 (PGE2), a prostaglandin that has physiological functions such as platelet aggregation.[1] COX has a heme at the active site and the redox state of the heme is crucial for cyclooxygenase and peroxidase activities of the enzyme.[2] However, COX is subject to self-inactivation, possibly because of the oxidation of a tyrosine residue at the heme active site of the enzyme.[3] Therefore, COX is liable to lose its activity by oxidative modification of the enzyme. COX-2 expression is reported to increase in inflammatory diseases such as rheumatoid arthritis (RA),[4, 5] and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS)[6] and Alzheimer disease (AD)[7] where the enzyme oxidatively crosslinks with β-amyloid.[8] Therefore, nonsteroidal anti-inflammatory drugs (NSAIDs), typical inhibitors of COX-2, may have beneficial effects on these diseases.[9–11] There is evidence that aberrant metabolism of Cu2+, a redox-active biometal that can cause the oxidation of proteins, may also play a pathogenic role in these conditions.[12–15] Herein, we report that COX-2 is specifically inactivated by the physiological concentration of Cu2+ ions, and discuss its relevance in a clinical situation. Table 1 shows the effects of various biometals on the production of PGE2 by COX-2 activity. We measured PGE2 production to reflect COX-2 activity, as PGE2 is the major end-product of the partially-purified enzyme,[17] and can be sensitively quantified by EIA. We did not find any production of PGE2 from the sample without COX-2 (data not shown). At 500 nM, only Cu2+ inhibited PGE2 production significantly (P< 0.01, t test) by% 50%. The ligand for Cu2+(3 μM glycine) itself caused no inhibitory effect on the enzyme (data not shown). The background levels of Cu2+ in our buffers is routinely measured at less than 20 nM,[18] which was considered negligible compared with the concentration of Cu2+ added in this study. Cu2+ inhibited COX-2 activity in a concentration-dependent manner (Figure 1). The IC50 for Cu2+ was% 500 nM, which is a stoichiometric ratio of 3.5: 1 (Cu2+: enzyme). On the other hand, in the presence of 10 μM EDTA, the inhibitory effect of Cu2+ at concentrations up to 2000 nM was abolished (Figure 1). The amount of PGE2 produced in the presence of EDTA without Cu2+ was less than that in the sample lacking EDTA. This might be explained by EDTA chelating some Fe on the heme from the active site of COX-2, so decreasing the activity of the enzyme.